Fed batch culture method for protein secreting cells

ABSTRACT

In a process of production of a useful protein by culturing a human embryonal kidney cell-derived 293 strain which is introduced with a useful protein-expressing gene, it becomes possible to obtain a culture fluid containing the useful protein in a high concentration using an extremely small amount of the medium, by carrying out a fed-batch culture and adding a sugar or a sugar and a calcium in a specified stage of the culture.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for the production of a usefulprotein by making cells secrete the useful protein. More particularly,this invention relates to a process which comprises culturing a humanembryonal kidney-derived 293 cell transformed so as to express a usefulprotein to obtain a culture fluid containing the desired useful proteinin a high concentration, and recovering the useful protein from theculture fluid.

2. Description of Related Art

Production of useful proteins, particularly physiologically activeproteins by culturing animal cells, is widely carried out. Particularly,processes of producing useful proteins by culturing animal cellstransformed so as to secrete the useful proteins are indispensabletechniques for production of certain useful proteins.

Animal cells are superior to bacteria and yeasts in the ability ofpost-translational modification. However, when animal cells are used,the cost of the medium and purification are relatively high due to a lowproduct concentration in culture fluid. Therefore, it is important forindustrial production to raise the concentration of useful proteinssecreted by animal cells. In a simple batch culture method,proliferation of the cells stops at some point of the culture periodmainly due to the exhaustion of nutrients, and secretion of the proteinalso stops. In order to improve this simple batch culture method, aso-called fed-batch culture method is carried out in which exhaustednutrients, for example sugars, amino acids or a fresh medium, are addedcontinuously or intermittently during the culture period.

In this specification, a "simple batch culture method" means a methodwhich comprises inoculating cells into a medium in a culture vessel tostart culture, and carrying out the culture without substantial additionof part or all of the nutrients or a new medium until completion of theculture, and a "fed-batch culture method" means a method comprisinginoculating cells into a medium in a culture vessel to start culture,and carrying out the culture while adding part or all of the nutrientsor a fresh medium continuously or intermittently into the culturevessel, without substantially taking out the culture fluid from theculture vessel.

It is known that in a fed-batch culture method, the culture period isprolonged and higher cell density and product (protein) concentrationcan be obtained compared to a simple batch culture method (J. B.Griffith, Animal Cell Culture and Production of Biologicals, pp.401-410, Klumer Academic Publishers, R. Sasaki and K. Ikura (eds.),(1991), S. Reuveny et al., J. Immunol. Methods, 86, 53 (1986), S.Reuveny et al., J. Immunol. Methods, 86, 61 (1986)). In the aboveliterature of J. B. Griffith, a comparison shown in the following Table1 is given as a typical example.

                  TABLE 1                                                         ______________________________________                                                Number of Product                                                     Culture cells     yield      Per liter                                                                              Length                                  type    (millions)                                                                              (mg/week)  (mg/month)                                                                             (days)                                  ______________________________________                                        Batch   3         100        200       7                                      Fed     6         200        500      14                                      batch                                                                         ______________________________________                                    

The above S. Reuveny et al. compares a simple batch culture with afed-batch culture in the latter of the above literatures. Thiscomparative experiment has been conducted under the followingconditions: (1) The simple batch culture comprised inoculating hybridomacells at a density of 3×10⁵ cells/ml in 100 ml of a medium and carryingout culture for 8 days, and (2) the fed-batch culture comprisedinoculating hybridoma cells at a density of 3×10⁵ cells/ml in 60 ml of amedium, and then carrying out the culture for 8 days while adding 6 mlof a fresh medium once a day after the density had become about 1×10⁶cells/ml 2 days after inoculation. The results show that the antibodyproductivities in both methods are 15 mg/l/day and 27 mg/l/day,respectively. Thus, S. Reuveny et al. teaches an about 1.8-fold increaseof the productivity (antibody) in the fed-batch culture compared to thesimple batch culture.

Thus, in a fed-batch culture method, cell density and proteinconcentration can be increased compared to a simple batch culturemethod. However, in view of medium costs, it is preferable that cellproliferation is rather suppressed at a certain stage and a highproductivity of protein is maintained for a long time. In a fed-batchculture method, much energy tends to be consumed for cell proliferation.Therefore, if it is possible to suppress cell proliferation after thesufficient cell growth and to direct energy to the production of thesubstance (protein), the protein concentration thereof in the culturefluid becomes further higher. However, an inexpensive and effectiveprocess has not been reported yet.

On the other hand, EP 0343635A2 discloses a process in which calcium ionconcentration in the culture fluid is maintained at a low concentrationfrom 0.002 to 0.3 mM in a process to culture an adherent animal cellline such as human embryonal kidney cell-derived 293 cell, thehamster-derived BHK cell, the hamster-derived CHO cell or a transformantthereof continuously in a serum-free medium in a suspension state. Thisprocess is a process to carry out continuous culture in which suchadherent animal cells are maintained in a suspension state bymaintaining the calcium ion concentration at the above low level, and isa so-called perfusion-suspension culture method which comprisescontinuously or intermittently supplying a fresh medium into the culturevessel and taking out the spent medium from the culture vessel. Thus inthis process, although it is possible to culture adherent cells at ahigh density and in a suspension state for an extremely long period, itis difficult to accumulate a useful protein secreted by the cells in ahigh concentration, and the medium cost to produce the useful proteintends to be considerably high.

SUMMARY OF THE INVENTION

The first object of this invention is to provide an improved process ofculturing a 293 strain transformed so as to secrete a desired usefulprotein in a suspension state according to a fed-batch culture method.

The second object of this invention is to provide a process of afed-batch culture method which comprises proliferating the cell strainat the initial stage, suppressing substantial proliferation of the cellsafter the cell density has reached a certain level, and thereafter,continuing the production of the useful protein by maintaining theculture for a long period.

The third object of this invention is to provide a process of afed-batch culture method in which medium consumption in the productionof a certain amount of the useful protein is reduced, namely a processwherein medium costs are saved.

The fourth object of this invention is to provide a process whichcomprises culturing the cell strain in a suspension fed-batch culturestably for a long period to obtain a culture fluid containing a usefulprotein accumulated in a high concentration.

Another object of this invention is to provide an industrial process toobtain a culture fluid containing a useful protein in a highconcentration such that the useful protein can be separated efficiently.

Still another object of this invention is to provide a culture processfor obtaining the culture fluid containing Protein C, activated ProteinC, or a physiologically active protein having similar activity to themin a high concentration.

Still other objects of this invention will become apparent from thefollowing description.

As a result of intensive research, the present inventors have found thatthe objects and advantages of this invention can be achieved by aprocess of production of a useful protein by culturing mammalian cell ina suspension state, wherein the improvement is in that the mammaliancell is a human embryonal kidney cell-derived 293 strain which isintroduced with a useful protein-expressing gene, and in that theprocess comprises

(a) starting the culture at a cell density of 5×10⁴ to 5×10⁵ cells/ml,

(b) adding a sugar in a culture vessel to give a concentration of 1 to 7g/l at any time after the cell density in the culture vessel has becomethree times higher than the inoculum cell density and when the celldensity is in a range of 5×10⁵ to 5×10⁶ cells/ml,

(c) continuing the culture until the substantial increase of theconcentration of the useful protein does not come to be recognized whilethe suppression of the substantial lowering of the pH of the culturefluid is maintained, and then stopping the culture, and

(d) taking out the culture fluid from the culture vessel and recoveringthe useful protein from the culture fluid.

In the process of this invention, at least 6 to 7-fold higher productconcentration compared with a simple batch culture without adding asugar can be accomplished by selecting a transformed 293 strain andadding a certain amount of a sugar in a certain stage of the culture.Further, even in comparison with a method in which a fresh medium isadded additionally in the middle of culture, as is usually put intopractice in a fed-batch culture method, a useful protein can be obtainedin a several fold larger amount in the process of this invention.

Although the reason is not clear, this phenomenon is surmised to beattributed to the inherent character of the 293 strain to the sugaradded in the middle of culture.

Further, another finding of the present inventors indicates that itbecomes possible to have the 293 cells secrete a further large amount ofuseful protein by starting the culture of the 293 strain in a mediumcontaining calcium ions in a low concentration and adding a calcium salttogether with a sugar in a certain stage of the culture. According tothis another finding, proliferation of the cells is substantiallysuppressed and secretion of the useful protein is further promoted bythe addition of the calcium. Therefore, it becomes possible to obtainthe useful protein in a larger amount.

Thus, according to another embodiment of this invention, there isprovided a process of production of a useful protein by culturingmammalian cell in a suspension state, wherein the improvement is in thatthe mammalian cell is a human embryonal kidney cell-derived 293 strainwhich is introduced with a useful protein-expressing gene, and in thatthe process comprises

(i) starting the culture in a culture medium having a calcium ionconcentration of 0.002 to 0.25 mM at a cell density of 5×10⁴ to 5×10⁵cells/ml,

(ii) proliferating the cells until the cell density in the culture fluidbecomes three times or still higher than the inoculum cell density andcomes to a range of 5×10⁵ to 5×10⁶ cells/ml,

(iii) adding a sugar and a calcium into the culture fluid so that theincrease of the cell density is substantially suppressed and theproduction of the useful protein is maintained,

(iv) continuing the culture until the substantial increase of theconcentration of the useful protein in the culture vessel does not cometo be recognized, and then stopping the culture, and

(v) taking out the culture fluid from the culture vessel and recoveringthe useful protein from the culture fluid.

The process of this invention is described more clearly below.

A cell line which is used in this invention is a transformant of thehuman embryonal kidney cell-derived 293 strain. This human embryonalkidney cell-derived 293 strain itself is known and, for example,deposited with ATCC under the accession number of CRL 1573, andtherefore, is a cell line easy to get. In the process of this invention,a transformant obtained by introducing a useful protein-expressing geneinto this 293 strain is cultured. This transformant of the 293 strain isa cell line wherein a gene was integrated so as to secrete an objectprotein. As useful object proteins, there can be mentioned proteinshaving various physiological properties. Useful proteins can be Glaproteins. Such Gla proteins are proteins having a Gla-domain, andspecifically, examples are Protein C, activated Protein C, the FactorVII, Factor IX, Factor X, prothrombin, osteocalcin, Protein S, ProteinZ, etc.

Particularly, the process of this invention is suitable for productionof human Protein C, activated human Protein C, proteins having aphysiological activity similar to them, or precursor proteins thereof.In the above, the precursor proteins mean ones which can be converted tohuman Protein C, activated human Protein C or proteins having aphysiological activity similar to them by processings such as partialconversion, cleavage or deletion of the proteins.

The 293 strain expressing human Protein C or activated human Protein Cis known per se, and the known transformants of the 293 strain aresuitable as a cell line applicable for the process of this invention(for example, refer to U.S. Pat. No. 4,968,626 and EP 319944A2).

In the process of this invention, the transformant of the 293 strainmentioned above is cultured in a suspension state by a fed-batch method.In this occasion, a culture vessel to be used can be a vessel used in ausual batch culture, for example, a tank-type vessel. This transformantof the 293 strain is an adherent cell line, and hence, in order tomaintain the cells in a suspension state in the culture vessel, theculture is carried out under stirring. Therefore, the culture is carriedout by a usual stirred culture method.

The culture vessel is equipped with oxygen supply means together withstirring means. Oxygen can be supplied by bubbling of oxygen gas or air,or using gas-permeable porous tube, or by any other suitable means. As aporous tube, a Teflon® tube or a silicone rubber tube is recommended.Oxygen is so supplied that dissolved oxygen can be maintained at about 2to 5 ppm, preferably about 3 ppm, in the culture fluid.

Many kinds of culture media can be used for the process of thisinvention. Synthetic media known well and used widely can be used asbasal media. As basal media, there can be mentioned RPMI-1640 (RPMI1640medium), MEM (Eagle's minimum essential medium), DME (Dulbecco'smodification of Eagle's medium), Isocove (Isocove's modification ofDulbecco's medium), 199 (199 medium), F10 (Ham's F10 medium), F12 (Ham'sF12 medium), etc. All these basal media are commercially available andeasy to get.

These media are used alone or in an appropriate combination of two ormore, and various amino acid, vitamins, inorganic salts and glucose canbe added additionally to make a modified medium.

In the process of this invention, a serum-free medium can be used, andthe use of it is desirable for lowering medium costs. Although mediacontaining serum can also be used, medium cost is generally high andhence, the advantage of addition of serum is small. Even when serum isadded, the amount is preferably at most 5 vol %, more preferably at most3 vol %.

It is industrially advantageous to carry out the process of thisinvention using a serum-free medium. In this occasion, "eRDF" medium canbe used as a basal medium. This eRDF medium is a medium obtained bymixing RPMI-1640 medium, Ham's F12 medium and Dulbecco's modification ofEagle's medium in a ratio of 2:1:1 and further adding glucose, aminoacids, etc., and is a medium known per se (refer to MonoclonalAntibodies: Production and Application, pp. 107-141, Alan R. Liss, Inc.1989, and Hiroki Murakami, "Serum-Free Media Used for Cultivation ofHybridomas").

The basal media mentioned above or the eRDF medium usually contain about0.3 mM or more, particularly about 0.5 mM to about 2 mM, of calcium ion.

In the process of this invention, it is desirable to add into the basalmedium, further, growth factors, for example, ITES (a mixture ofinsulin, transferrin, ethanolamine and sodium selenite).

As stated above, it is one of preferred embodiments of the process ofthis invention to start culture using a medium containing calcium ion ina low concentration. Such a medium containing calcium ion in a lowconcentration (hereafter sometimes referred to as "low Ca medium") meansa medium containing 0.0025 to 0.25 mM, preferably 0.05 to 0.2 mM ofcalcium ions.

Since the basal media mentioned above contain, in many cases, calciumion in a concentration higher than those of the low Ca media, a mediumcontaining no calcium ion or containing a calcium ion in an extremelylow content should be specially prepared for producing a low Ca mediumfor the preferred embodiment. Thus, it is desirable to use a mediumprepared using eRDF composition in a low calcium concentration as abasal medium.

In the fed-batch culture method of this invention, culture is started byinoculating a transformant of the 293 strain in a medium in a culturevessel. In this occasion, it is advantageous to inoculate the cells in adensity of the range of 5×10⁴ to 5×10⁵ cells/ml, preferably in a densityof the range of 8×10⁴ to 3×10⁵ cells/ml. When the inoculating celldensity is lower than the range above, it takes long time to reach adesired density and nutrients are considerably consumed before thedensity is gained, and consequently, it becomes difficult to obtain adesired useful protein efficiently. On the other hand, in the case thatan inoculum cell density is higher than the range above, it becomesnecessary to prepare a lot of cells before the culture is started, whichis economically disadvantageous.

Generally, when culture is started and continued, the cell densitygradually increases and, therewith, the concentration of the usefulprotein, too, increases, and after further continuation of the culture,the cell density reaches at a maximum value. The cells die after that,and the culture terminates.

In the process of this invention, a sugar is added in the culture fluidat any point of culture period when, after the start of culture, thecell density becomes three times or still higher than the inoculumdensity and reaches the range of 5×10⁵ to 5×10⁶ cells/ml. It isparticularly preferable to add the sugar at any point of culture periodwhen the cell density becomes four times or still higher than theinoculum cell density and when the cell density is in the range of 8×10⁵to 3×10⁶ cells/ml.

Further, it is desirable to determine the time of the addition of thesugar upon monitoring the cell density and the sugar concentration inthe culture fluid. Namely, it is advantageous to add a sugar between thetime when the sugar concentration in the culture fluid becomes 70% orless, preferably 50% or less, than the initial concentration and thetime when the sugar concentration becomes 0.2 g/l, preferably 0.3 g/l.

In the process of this invention, it is adequate to make the addition ofa sugar in the middle of culture in the range above upon monitoring thecell density and the sugar concentration. The amount of a sugar to beadded depends on the cell density and sugar concentration in the culturefluid at the time of addition, the culture period or calcium ionconcentration, etc. However, the sugar is generally so added that thesugar concentration in the culture fluid can become 1 to 7 g, preferably2 to 5 g, per liter of the culture fluid.

As the sugar to be added, there can be mentioned glucose, mannose orfructose, etc., but glucose or mannose is preferable, and glucose is themost desirable.

The number of the times of addition of the sugar can be once or twice ormore so long as the addition is made in the course of the time above.However, the advantage of repeated addition of a sugar is small, and theobject is fully attained by adding once.

A preferred embodiment of the process of this invention is to start aculture using the low calcium medium mentioned above, and, as statedabove, add a specific amount of a sugar into the culture fluid in themiddle of the culture and add a calcium salt.

In this case, it is advantageous to add a sugar and a calcium salt intothe culture fluid so that the cell density in the culture vessel iscontrolled in the range of ±50% (-50% to +50%) preferably in the rangeof ±30% (-30% to +30%) of the standard maximum cell density. By suchaddition of the sugar and calcium salt, the increase of cell density issubstantially suppressed and the culture period is remarkably prolongedwithout any decrease of the secretion of a useful protein. The "standardmaximum cell density" means the value of a maximum density which reachesin the culture fluid when a simple batch culture is carried out underthe same culture conditions as a fed-batch culture in which sugar andcalcium salt are added in the middle of culture, and it can easily bedetermined by a preliminary experiment.

The time of addition of the calcium may be in the range almost the sameas that of the sugar, but it is unnecessary to add the calcium togetherwith the sugar at the same time. Preferably, the calcium salt is addedin such an amount that the calcium ion concentration in the culturefluid becomes 0.3 to 3 mM at any time between the time when the sugar.concentration in the culture fluid becomes 50% or less of the sugarconcentration at the time of the start of culture and the time prior tothe lapse of two days after the addition of the sugar.

When a culture is, thus, started using a low calcium medium, and a sugarand a calcium are added in the middle of the culture, the increase ofcell density is substantially suppressed after the addition of them andproduction of a useful protein is maintained for a long period, andhence, a culture fluid containing the useful protein in a highconcentration can be obtained.

The addition of a calcium salt is advantageously conducted at any timebetween the time when the sugar concentration in the culture fluidbecomes 70% or less of the sugar concentration at the time of the startof culture and the time of addition of the sugar. It can be made withthe addition of the sugar at a time. It is still preferable to add thecalcium such that the calcium ion concentration in the culture fluidbecomes 0.5 to 2.5 mM.

Calcium salts to be added can be ones soluble in water, for example,calcium chloride (CaCl₂) or calcium nitrate (Ca(NO₃)₂).

Although the cells tend to aggregate and form clumps by the addition ofthe calcium salt, the size of the clumps is not so big as to causesubstantial hindrance against the maintenance of secretion of the usefulprotein, since the increase of the cell density is substantiallysuppressed.

Thus in the culture in the process of this invention, the pH of theculture fluid is maintained in the range of 6.5 to 7.8, preferably inthe range of 6.6 to 7.5.

It is desirable that culture is continued while monitoring theconcentration of the useful protein in the culture fluid and the cultureis stopped when the substantial increase of the concentration of theuseful protein cannot be recognized.

After the completion of the culture, the culture fluid is taken out fromthe vessel and the cells are separated and removed. For the separationof the cells, a means such as filtration or centrifugation is adopted.For the recovery of the useful protein from the culture fluid from whichthe cells have been removed, a suitable method is selected depending onthe kind of the useful protein. For example, immunological adsorption,ion exchange resin adsorption or the like is employed.

When the useful protein is human Protein C, activated human Protein C, aprotein having a physiological activity similar to that of them, or aprecursor protein thereof, the useful protein can, for example, berecovered by a separation method using a monoclonal antibody disclosedin U.S. Pat. No. 4,902,614, or a separation method using an ion exchangeresin disclosed in U.S. Pat. No. 4,981,852.

According to the process of this invention, it is possible to obtain aculture fluid containing a useful protein in a high concentration and todecrease extremely the amount of the medium consumed per the obtaineduseful protein by the culture of a transformant of the 293 strain. Thus,it is possible to lower the costs of the production of a useful proteinsignificantly since the consumption of an expensive medium is loweredand sugars and calcium salts to be added in the middle of culture areextremely inexpensive.

The following are a consumption of the medium per the unit amount (mg)of the useful protein in a perfusion continuous culture method, a simplebatch culture method, a conventional fed-batch culture method and thefed-batch culture method according to the invention.

Culture methods:

(A) A perfusion continuous culture method (EP 0343635A2)

(B) A simple batch culture method (a fresh medium, a sugar and a calciumsalt were not added at all in the middle of the culture)

(C) A conventional fed-batch culture method (a fresh medium was added inthe middle of culture)

(D) A fed-batch culture method of the invention (a sugar and a calciumsalt were added in the middle of the culture)

The premise for the calculation:

(1) Cell strain

The human Protein C-producing 293 strain used in Example 1

(2) Useful protein

Human Protein C (PC)

(3) Medium

ITES eRDF containing 0.1 mM calcium ions

(4) Volume of the culture vessel 350 ml

(5) Perfusion continuous culture method

It is assumed that after the PC concentration reaches maximum, theconcentration is maintained during the culture period (perfusion rate is300 ml/day).

(6) Culture period

6 months (in the case of batch culture method, the initial batch cultureis carried out until the PC concentration reaches maximum, and it isassumed that the batch is repeated taking the number of days needed forthe initial batch culture as the repeating unit.)

(7) Maximum PC concentration in the culture fluid and the number of daysof culture

As shown in the Table 2.

(8) Amount of the medium used and the amount of PC produced

As shown in the Table 3.

                  TABLE 2                                                         ______________________________________                                                                    Culture period                                                     Maximum PC needed to get                                                      concentration                                                                            the maximum PC                                                     in the fluid                                                                             concentration                                     Culture method   (μg/ml) (days)                                            ______________________________________                                        (A)  Perfusion con-  17.9        6                                                 tinuous culture                                                               method                                                                   (B)  Simple batch    9.0        11                                                 culture method                                                           (C)  Conventional fed-                                                                             11.0        7                                                 batch culture method                                                     (D)  Fed-batch       102.9      28                                                 culture method                                                                (this invention)                                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                                              Medium                                                        Amount   Amount used per                                                      of the   of     unit                                                Number of medium   PC     amount                                              repeat of used     produced                                                                             of PC                                   Culture method                                                                            culture   (ml)     (mg)   (ml/mg)                                 ______________________________________                                        (A)  Perfusion   1        53,800 958    56.2                                       continuous                                                                    culture                                                                       method                                                                   (B)  Simple batch                                                                             16        4,800  43.2   111.1                                      culture                                                                       method                                                                   (C)  Conventional                                                                             25        7,500  82.5   90.9                                       fed-batch                                                                     culture                                                                       method                                                                   (D)  Fed-batch   6        1,800  185.2  9.7                                        culture                                                                       method                                                                        (this                                                                         invention)                                                               ______________________________________                                    

As apparent from the Tables above, it is possible, according to theprocess of this invention, to produce a protein with an extremely smallamount of the medium. A usual fed-batch culture method (C) is generallya method of adding a fresh medium, but in the process of this invention,it is possible to lower the amount of the medium used more greatly thanthis method (C).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing the results of the culture of Example 1.

FIG. 2 is a drawing showing the results of the culture of ComparativeExample 1.

FIG. 3 is a drawing showing the results of the culture of ComparativeExample 2.

FIG. 4 is a drawing showing the results of the culture of Example 2.

FIG. 5 is a drawing showing the results of the culture of Example 3.

FIG. 6 is a drawing showing the results of the culture of ComparativeExample 3.

FIG. 7 is a drawing showing the results of the culture of ComparativeExample 4.

FIG. 8 is a drawing showing the results of the culture of ComparativeExample 5.

FIG. 9 is a drawing showing the results of the culture of ComparativeExample 6.

FIG. 10 is a drawing showing the outline of the preparation proceduresfor BHK/J3-26.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S) EXAMPLES

The process of this invention is specifically described below accordingto examples.

Example 1

1) Medium

As a basal medium, eRDF (a mixture of RPMI-1640, DME and F12 in a ratioof 2:1:1 enriched with amino acids, glucose, etc.) deprived of calciumsalts (calcium chloride and calcium nitrate) except calciumpantothenate, [hereafter referred to as low Ca eRDF] was used. Insulin,tranaferin, ethanolamine and sodium selenite (ITES) were added as growthfactors. The concentration of ITES are 9 μg/ml, 10 μg/ml, 10 μM and 20μM, respectively.

2) Culture method and results

The culture medium was put in a 350-ml culture vesselautoclave-sterilized in advance so that the net culture volume becameabout 300 ml, and therein was inoculated a Protein C-producing strain293/21-26 obtained by introducing a gene encoding the amino acidsequence of human Protein C into the 293 strain derived from a humanembryonal kidney cell obtained from ATCC using the method disclosed inWO92/13079.

Oxygen gas was introduced into the gaseous phase above the culture fluidin the culture vessel to maintain the dissolved oxygen concentration at3 ppm.

The culture fluid in the culture vessel was held at 37° C. The culturevessel was equipped with a marine type agitating impeller and theagitation speed was 40 rpm.

Eight days after the culture started, 1.0 g of D-glucose and 32.6 mg ofcalcium chloride dihydrate were added in the form of 3 ml of a mixedaqueous solution (the concentration of each component in the culturefluid was 19 mM for glucose and 0.74 mM for calcium chloride).

Culture results are shown in FIG. 1. In FIG. 1, PC denotes human ProteinC (this applies to the following Examples and Comparative Examples). Thecell line, the medium and the additives in the middle of the cultureused in this example are as follows.

Cell: 293/21-26

Medium: 0.1 mM CaCl₂ +low Ca eRDF+ITES

Additives in the middle of the culture:

1 g of glucose+32.6 mg of calcium chloride dihydrate (3 ml of a mixedaqueous solution)

Comparative Example 1

The same operations as in Example 1 were carried out except that anyaddition in the middle of the culture was not made. Culture results areshown in FIG. 2. The cell line, the medium and the additives in themiddle of the culture used in this example are as follows.

Cell: 293/21-26

Medium: 0.1 mM CaCl₂ +low Ca eRDF+ITES

Additives in the middle of the culture: None

Comparative Example 2

The same operations as in Comparative Example 1 were carried out exceptthat eRDF (calcium ion concentration 0.74 mM) was used as a basalmedium. Culture results are shown in FIG. 3. The cell line, the mediumand the additives in the middle of the culture used in this example areas follows.

Cell: 293/21-26

Medium: eRDF+ITES

Additives in the middle of the culture: None

Example 2

The same operations as in Example 1 were carried out except that onlyglucose was used as an additive in the middle of the culture. Cultureresults are shown in FIG. 4. The cell line, the medium and the additivein the middle of the culture used in this example are as follows.

Cell: 293/21-26

Medium: 0.1 mM CaCl₂ +low Ca eRDF+ITES

Additives in the middle of the culture:

1 g of glucose (3 ml of an aqueous solution)

Example 3

The same operations as in Comparative Example 3 were carried out exceptthat eRDF was used as a basal medium. Culture results are shown in FIG.5. The cell line, the medium and the additives in the middle of theculture used in this example are as follows.

Cell: 293/21-26

Medium: eRDF+ITES

Additives in the middle of the culture:

1 g of glucose (3 ml of an aqueous solution)

Comparative Example 3

The same operations as in Example 1 were carried out except that onlycalcium chloride was used as an additives in the middle of the culture.Culture results are shown in FIG. 6. The cell line, the medium and theadditive in the middle used in this comparative example are as follows.

Cell: 293/21-26

Medium: 0.1 mM CaCl₂ +low Ca eRDF+ITES

Additives in the middle of the culture:

32.6 g of calcium chloride dihydrate (3 ml of an aqueous solution)

Comparative Example 4

The same operations as in Comparative Example 1 were carried out exceptthat the initial culture volume was 150 ml, and a fresh medium was fedat a constant speed (6.25 ml/hr) over a period from the fourth day tothe fifth day of the culture to make the culture volume 300 ml. Theresults are shown in FIG. 7.

The results of Examples 1 to 3 and Comparative Examples 1 to 4 aresummarized together in the following Table 4.

                  TABLE 4                                                         ______________________________________                                        Calcium con-             Maximum   Maximum                                    concentration in         cell density                                                                            PC con-                                    the initial              (×10.sup.6                                                                        centration                                 medium (mM)   Additive   cells/ml) (μg/ml)                                 ______________________________________                                        Ex. 1 0.10        glucose +  2.4     103                                                        calcium                                                                       chloride                                                    Comp. 0.10        none       2.6     9.0                                      Ex. 1                                                                         Comp. 0.74        none       4.3     15.3                                     Ex. 2                                                                         Ex. 2 0.10        glucose    6.3     54.8                                     Ex. 3 0.74        glucose    6.3     55.0                                     Comp. 0.10        calcium    5.4     15.3                                     Ex. 3             chloride                                                    Comp. 0.10        fresh      2.3     10.1                                     Ex. 4             medium                                                      ______________________________________                                    

Comparative Example 5

The same operations as in Comparative Example 1 were carried out exceptthat a human Protein C-producing cell line BHK/J3-26 strain was used inplace of the 293/21-26. This BHK/J3-26 is a cell line obtained byintroducing a gene encoding the amino acid sequence of human Protein Cinto the BHK cell strain according to the method described below. Theresults are shown in FIG. 8.

Comparative Example 6

The same operations as in Example 1 were carried out except that theBHK/J3-26 cell line mentioned in Comparative Example 5 was used in placeof the 293/21-26, and the following additive in the middle of theculture was used. The results are shown in FIG. 9.

0.5 g of glucose+32.6 mg of CaCl₂.2H₂ O (3 ml of a mixed aqueoussolution)

In the above Examples and Comparative Examples, 293/21-26 and BHK/J3-26,respectively, were prepared by following methods.

(A) Preparation of 293/21-26

293 Strain was transformed with plasmid TZm5-PC9002 having DNA sequenceencoding amino acid sequence of human protein C described in page 25 ofWO 92/13079 according to the method described also in the WO 92/13079 toobtain human protein C-producing cell line, 293/21-26.

(B) Preparation of BHK/J3-26

Plasmid TZm1D-9002 having DNA sequence encoding amino acid sequence ofhuman protein C was inserted into BHK strain, and plasmid ZmB4 KEX2having DNA sequence encoding amino acid sequence of KEX2 (preparationmethod is described below) and the above TZm5-PC9002 were co-transfectedto obtain BHK/J3-26.

Methods of preparing TZm1D-9002 and ZmB4 KEX2

TZm1D-9002 and ZmB4 KEX2 were prepared from ZmB3 KEX2 and ZmB4 1058(preparation methods are described below) by the following procedures.The procedures are outlined in FIG. 10.

ZmB3 KEX2 was digested with SfiI and NdeI, and KEX2 gene-containing DNAfragment of 4.31 Kbp was isolated (K-fragment). Further, ZmB4 1058 wasdigested with ApaI, SfiI and NdeI, and DHFR gene-containing DNA fragmentof 3.46 Kbp was isolated (D-fragment). K-fragment and D-fragment wereligated to obtain ZmB4 KEX2.

Further, the TZm1D-9002 was digested with SfiI and NdeI, and humanprotein C-containing DNA fragment of 5.98 Kbp was isolated (P-fragment).P-fragment and D-fragment were ligated to obtain TZm1D-9002.

Method of preparing ZmB3 KEX2

Plasmid KEX/Zem 229 described in EP 0319944A2 was digested with Bam HI,and KEX2 gene-containing DNA fragment was isolated (D'-fragment).Further, plasmid ZmB3 described in WO91/09960 was digested with Bam HI,and the resultant fragment and D'-fragment were ligated to obtain ZmB3KEX2.

Method of preparing ZmB3 1058

pDX/PC1058 described in EP 0266190 was digested with EcoRI, and humanprotein C-containing DNA fragment was isolated (P'-fragment). Further,plasmid PC962/ZMB-4 described in WO 91/09960 was digested with EcoRI,and DNA fragment not containing human protein C-encoding DNA wasisolated (Z-fragment). P'-fragment and Z-fragment were ligated to obtainZmB4 1058.

We claim:
 1. In a process for the production of a protein by culturingmammalian cells in a suspension state, the improvement comprisingculturing as the mammalian cells a human embryonal kidney cell-derived293 strain which is transformed with a gene encoding the protein, andthe improvement further comprising culturing the mammalian cell using aprocess which comprises(a) starting the culture of the mammalian cellsin a culture medium contained in a culture vessel at a cell density of5×10⁴ to 5×10⁵ cells/ml, (b) adding a sugar to the culture vessel togive a sugar concentration of 1 to 7 g/l at any time after the celldensity of the mammalian cells in the culture vessel has become threetimes higher than the starting cell density and when the cell density isin a range of 5×10⁵ to 5×10⁶ cells/ml, (c) continuing the culture untilthe concentration of the protein is no longer substantially increasing,while maintaining the pH of the culture fluid, and then stopping theculture, and (d) taking out culture fluid from the culture vessel andrecovering the protein from the culture fluid.
 2. The process accordingto claim 1 wherein the culture is started in the culture medium having acalcium ion concentration of 0.002 to 0.25 mM.
 3. The process accordingto claim 1 wherein the pH of the culture fluid is maintained in a rangeof 6.5 to 7.8.
 4. The process according to claim 1 wherein the sugar isadded to give a concentration of 2 to 5 g/l.
 5. The process according toclaim 1 wherein the sugar is added at any time between the time when thesugar concentration in the culture fluid is 50% or less of the sugarconcentration at the start of the culture and the time when the sugarconcentration in the culture fluid is 0.2 g/l.
 6. The process accordingto claim 2 wherein calcium is added in such an amount that the calciumion concentration in the culture fluid becomes 0.3 to 4 mM at any timebetween the time when the sugar concentration in the culture fluid is50% or less of the sugar concentration at the start of culture and thetime prior to the lapse of two days after the addition of the sugar. 7.The process according to claim 1 wherein the sugar is at least onemember selected from the group consisting of glucose, mannose andfructose.
 8. The process according to claim 1 wherein the culture iscarried out in a serum-free medium.
 9. The process according to claim 8wherein the serum-free medium is prepared using eRDF (enriched RDF)medium as a basal medium.
 10. The process according to claim 1 whereinthe protein is a Gla protein.
 11. The process according to claim 1wherein the protein is human Protein C or activated human Protein C. 12.The process according to claim 1 wherein no additional fresh medium isadded into the culture vessel during the culture.
 13. In a process forthe production of a protein by culturing mammalian cells in a suspensionstate, the improvement comprising culturing as the mammalian cells ahuman embryonal kidney cell-derived 293 strain which is transformed witha gene encoding the protein, and the improvement further comprisingculturing the mammalian cells using a process which comprises(i)starting the culture of the mammalian cells in a culture mediumcontained in a culture vessel having a calcium ion concentration of0.002 to 0.25 mM at a cell density of 5×10⁴ to 5×10⁵ cells/ml, (ii)proliferating the cells until the cell density in a culture fluidbecomes three times or higher than the starting cell density in a rangeof 5×10⁵ to 5×10⁶ cells/ml, (iii) adding a sugar and calcium to theculture fluid so that an increase of the cell density is substantiallysuppressed and production of the protein is maintained, (iv) continuingthe culture until the concentration of the protein in the culture vesselis no longer substantially increasing, and then stopping the culture,and (v) taking out the culture fluid from the culture vessel andrecovering the protein from the culture fluid.
 14. The process accordingto claim 13 wherein the addition of the sugar and the calcium to theculture vessel is carried out so that the cell density in the culturevessel is inhibited to be in the range of ±50% of the standard maximumcell density.
 15. The process according to claim 13 wherein the pH ofthe culture fluid is maintained in the range of 6.5 to 7.8.
 16. Theprocess according to claim 13 wherein the sugar is added to give aconcentration of 1 to 7 g/l, preferably 2 to 5 g/l.
 17. The processaccording to claim 13 wherein the sugar is added at any time between thetime when the sugar concentration in the culture fluid is 50% or less ofthe sugar concentration at the start of the culture and the time whenthe sugar concentration in the culture fluid is 0.2 g/l.
 18. The processaccording to claim 13 wherein the calcium is added in an amount suchthat the calcium ion concentration in the culture vessel becomes in therange of 0.3 to 4 mM at any time between the time when the sugarconcentration in the culture vessel becomes 50% or less of the sugarconcentration at the time of the start of culture and the time prior tothe lapse of two days after the addition of the sugar.
 19. The processaccording to claim 13 wherein the sugar is at least one member selectedfrom the group consisting of glucose, mannose and fructose.
 20. Theprocess according to claim 13 wherein the culture is carried out in aserum-free medium.
 21. The process according to claim 20 wherein theserum-free medium is the one prepared using eRDF (enriched RDF) mediumas a basal medium.
 22. The process according to claim 13 wherein theuseful protein is a Gla protein.
 23. The process according to claim 13wherein the useful protein is human Protein C, activated human ProteinC, a protein having the similar physiological activity to them, or aprecursor protein thereof.
 24. The process according to claim 13 whereina fresh medium is not additionally added into the culture vessel duringthe culture.